Differentiation of Trypanosoma brucei: the master regulator RBP10 and its targets

非洲锥虫是寄生的单细胞鞭毛虫。在哺乳动物宿主体内，繁殖的细长血流形态（在37°C时）转化为不分裂的短小粗壮形态。在被采采蝇吸收后，矮秆形式转化为分裂的原环锥体，然后是副环锥体，然后是哺乳动物感染的亚环形式。在培养中，在加入3mm顺式酸盐和27°C的温度变化后，粗大的血流形式转化为顺环形式。值得注意的是，锥虫并不控制单个蛋白质编码基因的转录。转录是多反式的，单个mrna在加工过程中被切除。基因表达受影响mRNA加工、翻译和衰变的rna结合蛋白控制。RNA结合蛋白RBP10在细长的血流形态中表达，对其存活至关重要，但在粗壮的血流形态和顺环细胞中不存在。RNAi、诱导表达、pull-down、RNA-Seq和tethering实验结果表明，RBP10在mRNA的3'-非翻译区与UA(U)6序列的mRNA结合，导致翻译抑制和mRNA破坏。RBP10靶点包括编码前环能量代谢酶、主要的前环表面蛋白、3种蛋白激酶、1种蛋白磷酸酶和3种前环特异性锌指rna结合蛋白ZC3H20、ZC3H21和ZC3H22的mrna。在血液形式中RBP10的消耗，并转移到27°C，导致转化为没有顺性的顺环形式。相反，在诱导或短暂表达RBP10的顺环形式并转移到37°C后，一些细胞转化为血流形式。（没有RBP10，它们就会死亡）结果表明，RBP10的表达和37°C的温度决定了血流型锥虫的身份。我们打算研究RBP10自身的表达是如何被控制的，以及下游靶点的功能。我们将首先比较已建立的和新分离的锥虫系的分化。如果新的锥虫能更有效地分化，将被用作实验模型。为了研究RBP10的表达是如何被控制的，我们将定义所需的RNA序列。为了寻找候选的调控蛋白，我们将首先鉴定所有处于不同生命周期阶段的mrna结合蛋白，然后将这些信息与现有数据集结合，如果可行的话，使用RNA亲和纯化。然后测试功能。我们将研究RBP10的磷酸化和周转在其功能和调控中的作用。为了跟踪RBP10下游的分化级联，我们将分析被RBP10抑制的潜在调节因子的功能：激酶和磷酸酶，以及三种锌指蛋白。到项目结束时，这些结果与补充工作的结果相结合，应该产生对锥虫发育阶段如何维持以及发育开关如何受到影响的机制理解。

African trypanosomes are parasitic unicellular flagellates. In a mammalian host, multiplying long slender bloodstream forms (at 37°C) convert to non-dividing short stumpy forms. After uptake into Tsetse, stumpy forms convert to dividing procyclic trypomastigotes, then epimastigotes, then mammalian-infective metacyclic forms. In culture, stumpy bloodstream forms convert to procyclic forms after addition of 3 mM cis-aconitate and a 27°C temperature shift.Remarkably, trypanosomes do not control transcription of individual protein-coding genes. Transcription is polycistronic and individual mRNAs are excised by processing. Gene expression is controlled by RNA-binding proteins that influence mRNA processing, translation, and decay. The RNA binding protein RBP10 is expressed in, and essential for survival of, long slender bloodstream forms, but is absent in stumpy forms and procyclics. Results from RNAi, induced expression, pull-down, RNA-Seq and tethering assays showed that RBP10 binds to mRNAs with the sequence UA(U)6 in their 3'-untranslated regions, causing translation repression and mRNA destruction. RBP10 targets include mRNAs encoding enzymes of procyclic energy metabolism, the major procyclic surface protein, 3 protein kinases, a protein phosphatase, and three procyclic-specific zinc finger RNA-binding proteins, ZC3H20, ZC3H21 and ZC3H22. Depletion of RBP10 in bloodstream forms, and transfer to 27°C, results in transformation to procyclic forms without cis-aconitate. Conversely, after induced or transient expression of RBP10 in procyclic forms and transfer to 37°C, some cells convert to bloodstream forms. (Without RBP10, they die.) together the results suggest that expression of RBP10 and a temperature of 37°C define bloodstream-form trypanosome identity.We here propose to investigate how expression of RBP10 itself is controlled, and the functions of downstream targets. We will first compare differentiation of established and freshly isolated trypanosome lines. New trypanosomes will be used as an experimental model if they differentiate more efficiently. To investigate how RBP10 expression is controlled we will define the RNA sequences required. To find candidate regulatory proteins, we will first identify all mRNA-binding proteins at different life-cycle stages, then combine the information with existing datasets, and use RNA affinity purification if feasible. Function will then be tested. We will investigate the roles of RBP10 phosphorylation and turnover in its function and regulation. To follow the differentiation cascade downstream of RBP10, we will analyse the functions of potential regulators whose expression is repressed by RBP10: the kinases and phosphatase, and the three zinc finger proteins.By the end of the project, the results, combined with those from complementary work, should yield a mechanistic understanding of how trypanosome developmental stages are maintained and how developmental switches are effected.